SUMMARY OF WORK This research area involves a model of in vitro differentiation of cardiomyocytes originating from embryonic stem cells (R1) and embryonic carcinoma cells (P19). The research is aimed at understanding the structure-function relationships of the sarcoplasmic reticulum calcium release channel and its relationship to spark formation and calcium movements in developing myocardial cells. We have been able to successfully differentiate pluriopotent ES and EC cells into embryoid bodies containing contracting cardiac-like cells. To identify, atrial versus ventricular like cells, expression vector constructs have been constructed that link ventricular markers to the green florescent protein (GFP), including the 2.1kb MLC2v-GFP construct. This construct has been shown to work very well after transient transfections, and thus were introduced into ES cells and neomycin resistance clones selected. Immunofluorescence analyses of the differentiating ES-derived cardiac cells using monoclonal and polyclonal antibodies have also been successfully performed for sarcomeric actins, troponin T; specific work on identifying the earliest stages of ryanodine receptor, SR CaATPase, phospholamban and dihydropyridine receptor expression are well under way. We have also established several techniques to measure the mRNA contents of these proteins, all of which are being used to develop a molecular model for the development of EC coupling and relaxation in early and late differentiating ES cells. Subsequently, we have developed a number of clones to the mouse ryanodine receptor 2 gene which have been modified for introduction into the R1 ES cells. Following positive-negative selection we have identified some clones which have undergone homologous recombination at the appropriate gene locus. These cells are now being characterized for in vitro analysis of the development of EC coupling in cardiomyocytes. This work is being performed in collaboration with the Excitation Contraction Coupling Unit of the Laboratory of Cardiovascular Science who have performed a number of very preliminary studies using the techniques of voltage clamping and confocal microscopy to analyze calcium spark formation, calcium transients and function of calcium handling proteins.